3 isobutyl 1 methylxanthine ibmx

Transvaginal ultrasound ovum pick up (OPU) was performed as previously reported [14 (link), 16 (link)]. Briefly, an Aloka real-time B-mode ultrasound system (Aloka SSD-4000, Hitachi Aloka Medical Ltd., Tokyo, Japan) and a 7.5 MHz electronic convex transducer (Hitachi Aloka Medical Ltd., Tokyo, Japan) were used to visualize the ovaries (S1 Fig). Visible follicles ≥ 3 mm in diameter were punctured using a disposable 20G x 2 ¾” needle (0.9 x 70 mm, Terumo, Eschborn, Germany). A vacuum pump (Aspirator 3, Labotect GmbH, Göttingen, Germany), adjusted to a negative pressure of 60 mmHg (20 ml/min) was used to recover follicular fluid. After puncture of four to five follicles, the oocyte collection system was flushed with Dulbecco’s PBS medium (AppliChem, Darmstadt, Germany), supplemented with 2.2 IU/ml heparin (AppliChem), 1% newborn calf serum (NBCS; PPA Laboratories, Coelbe, Germany), 6 μg/ml penicillin G (AppliChem) and 50 μg/ml streptomycin sulphate (AppliChem) for the standard protocol (TCM24) or additionally supplemented with 3-isobutyl-1-methylxanthine (IBMX, 500 μM, Sigma-Aldrich) for the protocol using the cAMP regulators (cAMP30) and dimethyl sulfoxide (DMSO, 46.6 mM, Sigma-Aldrich) as vehicle control (DMSO30). The vehicle control was necessary because IBMX was dissolved in DMSO. The interval from ovum pick up to oocyte searching did not exceed 20 min.

NF-κB p-p65 monoclonal antibody (93H1) was from Cell Signaling Tech (Danvers, MA, United States), and PDE4 polyclonal antibody (ab14628) was from Abcam (Cambridge, United Kingdom). The BIOMOL Cyclic Nucleotide PDE Assay kit was from ENZO Life Sciences, Inc. (Farmingdale, NY, United States). LPS (Escherichia coli 0111:B4) and 3-isobutyl-1-methylxanthine (IBMX) were from Sigma-Aldrich (St. Louis, MO, United States), and rolipram was a gift from MedChem Express (Monmouth Junction, NJ, United States).
Immunohistochemistry staining of reconstituted psoriatic and normal tissue models was conducted using deparaffinized sections subjected to antigen retrieval, followed by incubation with anti-PDE4 primary antibody (1:100). Bound antibody was detected by DAB staining using a Ventana universal secondary antibody (Ventana Medical System, Tuscan, AZ, United States). All stained slides were digitally imaged at magnification equivalent to 20×. Random fields were scored by an independent pathologist.

HC (#H0888), human tumor necrosis factor-α (TNF-α, #8916), lipopolysaccharide (LPS, #L4391) from Escherichia coli O111:B4, phorbol 12-myristate 13-acetate (PMA, #P8139), insulin (#I3536), isopropanol (#9516), and 3-isobutyl-1-methylxanthine (IBMX, #5879) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s phosphate-buffered saline (DPBS) without calcium and magnesium (#14190144) was purchased from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Cignal reporter assay kits for the NF-κB (#CCS-013 L) and GRE (#CCS-006 L) pathways were purchased from Qiagen (Hilden, Germany) and the DualGlo luciferase reporter system kit (#E1960) was procured from Promega (Madison, WI, USA). Genjet™ in vitro DNA transfection reagent (Ver. II, #SL100489) was purchased from (Rockville, MD, USA).
Polyclonal rabbit anti-human glucocorticoid receptor (#ab3578, 1 mg/mL; Abcam, Cambridge, UK) and monoclonal rabbit anti-human NF-κB p65 (#C22B4; Cell Signaling Technology, Danvers, MA, USA) antibodies were used for the immunofluorescence assay. Alexa 594 goat anti-rabbit antibody (#ab150080, Abcam) was used as the secondary antibody and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, #D9542; Sigma-Aldrich) was used to counter-stain DNA.

Small intestinal organoid generation from mouse jejunum (section taken 8–13 cm from the gastric pylorus), maintenance, and two-dimensional (2D) culture were carried out as previously described [50 (link)]. GIP secretion experiments were performed using 2D organoid-derived cultures, 18–24 h after seeding cells onto 48-well plates. Cells were washed three times in warm saline buffer (138 mM NaCl, 4.5 mM KCl, 4.2 mM NaHCO₃, 1.2 mM NaH₂PO₄, 2.6 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES; adjusted to pH 7.4 with 1 M NaOH, and supplemented on the day of experiment with 1 mM glucose and 0.1% fatty acid-free bovine serum albumin) before incubation in saline buffer for 30 min at 37 °C. The buffer was then completely removed before test reagents, dissolved in 150 µL saline buffer, were added to the organoid cultures. The test reagents used were 100 ng/mL LPS (Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL recombinant human IL-1Ra (PeproTech, Rocky Hill, NJ, USA), 10 ng/mL recombinant human MCP-1 (Peprotech), or 10 µM forskolin plus 10 µM 3-isobutyl-1-methylxanthine (IBMX) plus 10 mM glucose (all Sigma-Aldrich, St. Louis, MO, USA). After incubation for 2 h at 37 °C, supernatants were removed and centrifuged at 2000× g for 5 min at 4 °C, transferred to a fresh tube, and snap-frozen on dry ice.

4 to 8-week-old female, outbred, CD1 mice (Charles River; RRID:IMSR_CRL:22) were used. All animals were handled in accordance with ethics approved by the UK Home Office Animals (Scientific Procedures) Act 1986. GV oocytes were collected from ovaries punctured with a sterile needle; oocytes were stripped of their cumulus cells mechanically using a pipette. For bench handling, microinjections, and imaging experiments, oocytes were cultured at 37°C in medium M2 (Sigma), with the addition of 30 nM 3-isobutyl-1-methylxanthine (IBMX; Sigma) to arrest oocytes at prophase I where necessary. Data was only collected from oocytes which underwent GVBD with normal timings and had a diameter within 95-105% of the population average. To ensure reproducibility, oocyte data sets were gathered from a minimum of 3 independent experiments. For each independent experiment both control and treatments groups were derived from the same pool of oocytes collected from a minimum of 2 animals. Oocytes were selected at random for microinjection, however the investigators were not blinded to allocation during experiments or outcome assessment.

Carbamylcholine chloride (carbachol, CCh), Oxotremorine M (Oxo M), forskolin, 3-isobutyl-1-methylxanthine (IBMX), aldicarb, and standard chemicals were purchased from Sigma Aldrich. Clozapine-N-Oxide (CNO) was obtained from the National Institutes of Health (Bethesda, MD) as part of the Rapid Access to Investigative Drug Program funded by the National Institute of Neurological Disorders and Stroke. Substances applied were dissolved in the media or buffer the according experiment was performed in.

A549 (ATCC CCL-185) adenocarcinomic human alveolar basal epithelial cells (A549 cells) and 3T3-L1 (ATCC CL-173) primary murine fibroblast-derived adipocytes (3T3-L1 cells) were grown in Dulbecco’s modified Eagle medium (DMEM [Gibco] plus 4.5 g/liter d-glucose and pyruvate) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) (72 (link), 73 (link)). MG-63 (ATCC CRL-1427) osteosarcoma cells (MG-63 cells) were grown in minimum essential medium (MEM [Gibco] plus Earl’s salts and l-glutamine) with 10% FBS (74 (link)). Cells were incubated at 37°C and 5% CO₂ and maintained at ≤90% confluence for A549 and MG-63 cells and ≤80% confluence for 3T3-L1 cells. Differentiation of 3T3-L1 cells was performed as described before (75 (link)), with the following adjustments. The day after seeding, differentiation was induced by adding medium supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO, USA), 1 μg/ml insulin (Sigma-Aldrich), 1 μM rosiglitazone (Sigma-Aldrich), and 1 μM dexamethasone (Sigma-Aldrich). Two days after induction, the medium was changed to contain only 1 μg/ml insulin. Four days after induction, normal DMEM was used again, and the presence of lipid droplets was confirmed by light microscopy. The maximal passage numbers used were 23, 21, and 10 for A549, MG-63, and 3T3-L1 cells, respectively.